Our interest is to understand how the DNA replication frequency is adjusted in the cell cycle. Our system is plasmid P1 which belongs to a family of replicons commonly found in bacterial plasmids whose replication frequency is controlled by short repeating DNA sequences, called iterons.Mechanisms of Strand Opening: importance of DnaA box positionsStrand opening is a crucial step in the initiation of DNA replication. Since DNA replication is normally controlled at the stage of initiation, our premise is that steps leading to origin opening are important for controlling replication. Origin opening in plasmid P1 requires participation of host initiator DnaA, a DNA architectural protein HU, and a plasmid-encoded initiator, RepA. The DnaA protein has specific binding sites, the DnaA boxes, in the origin of E. coli, oriC,and of several plasmids including the P1 plasmid, but the requirements of DnaA boxes are different for the two origins. Whereas oriCrequires multiple boxes at invariant positions, a single consensus box at either end of the P1 orisuffices for the origin function. By probing with KMnO4, we found that the efficiency of strand opening in P1 ori depended on the disposition and number of DnaA boxes, but the location of opening remained the same regardless of their disposition. The boxes at the upstream end of the origin were more efficient in strand opening than those at the downstream end. A maximal efficiency in opening was seen when the boxes flanked both ends of the ori. Small changes in box positions at either end of P1 orireduced the efficiency of opening significantly, implying a critical role of box positions in the opening. It appears that, unlike the situation in oriC,DnaA can perform opening from alternative positions in P1 ori,albeit at varying efficiencies, but the task must be the same since the location of opening remains invariant.Mechanisms of Strand Opening: role of HUIn addition to two initiators, DnaA and RepA, that bind to P1 oriat specific sites, origin opening requires a third protein, HU, which is generally known as a non-specific DNA binding protein. Recent studies from unrelated systems have indicated that HU helps to form higher order structures by site-specific binding. We have found that HU has higher affinity for P1 oricompared to nonspecific DNA suggesting that HU may bind to P1 orisite-specifically. The evidence for site-spesific binding is being obtained by footprinting studies in vivoand in vitro. Together with the knowledge of binding sites for DnaA and RepA it may be possible to understand the origin topology that allows strand-opening.Regulation of Replication Frequency Iterons are believed to control replication frequency either by titrating replication initiator RepA or by causing steric hindrance to origin activity by RepA-mediated coupling of origins. The effects of titration and coupling are expected to increase with increase of origin concentration. We have developed a new strategy to examine the role of increased origin concentration on the replication frequency. The assay involves comparison of copy numbers of isogenic plasmid monomer and dimer. Our premise is that communication (coupling) would occur more readily when the two origins are in cis, as in a dimer, because of higher local concentration of one site in the vicinity of another, than when they are only in trans as in monomers. Dimer copy number was more than two-fold lower compared to monomer in support of the coupling model. However, the copy number difference could be partially reduced by providing extra initiators in trans, in support of the titration model. It appears that unsaturated origins are more proficient in coupling. Saturation of origins with initiators thwart the inhibitory activity of coupling and allow initiation. However, initiator excess does not cause over-initiation possibly by a separate mechanism as discussed below.We have found that transcription of the plasmid initiator gene is activated by replication. The promoter of the initiator gene maps within the iterons and initiator binding to them represses the promoter almost totally. We propose that this autoregulated promoter can only be transcribed when the passage of the replication fork cleans the promoter of the initiators but the burst of transcriptional activity produces, in addition to the initiator, an inhibitor that restrains reinitiation during the period of transient initiator excess. This would ensure initiator availability in spite of the low basal level. Our immediate goal is to identify the inhibitor and establish its physiological role.A Quantitative Model of Plasmid Replication FrequencyWe have developed a stochastic model of low-copy-number plasmid replication dependent upon a single function describing the probability of plasmid replication with cell age (i.e. a transition function). This function can be estimated directly from experimental data. As more experimental information about the various actors in initiator titration and other suggested models become available, the quantitative model will then be expanded to include more molecular details. This project is a collaborative effort with a theoretician, Paul Morrison, of NCRR NIH.,